Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
  • C12Q1/045—Culture media therefor

Definitions

• the invention relates to a method for the identification of at least two groups of microorganisms expressing the same enzymatic activity.
• the invention also relates to a particular reaction medium and its use for the identification of at least two groups of microorganisms expressing the same enzymatic activity.
• enzymatic substrates have been used to enable the determination of the presence or absence of enzymatic activities characteristic of microorganisms.
• These enzymatic substrates are generally composed of two parts, a first specific part of the enzymatic activity to be revealed, also called target part, and a second part serving as a marker, called a marker part, inducing for example a particular coloration of the colony. during the hydrolysis of the substrate, or the appearance of an easily detectable precipitate.
• strains of Escherichia coli are often evidenced by the revelation of an enzymatic activity of the osidase type such as ⁇ -glucuronidase or ⁇ -galactosidase activity.
• the genus Listeria can be detected by demonstrating ⁇ -glucosidase activity.
• an esterase activity especially for the detection of the genus Salmonella.
• the genus Salmonella has nonspecific esterases capable of hydrolyzing chromogenic synthetic substrates, for example indigogenic.
• the detection and / or identification of these bacteria is conventionally carried out on agar media, which allow the detection and / or identification suspicious colonies of esterase-active bacteria.
• a single enzymatic activity is not always sufficient to characterize a particular group of microorganisms of another group of microorganisms. For example, if you want to differentiate Gram-positive bacteria and Gram-negative bacteria, such as bacteria from the KES group ( Klebsiella , Enterobacter and Serratia , Gram-negative bacteria), and those from the genus Enterococcus (Gram-positive bacteria) ), it is necessary to detect several enzymatic activities to increase the specificity.
• the invention proposes to solve the problems of the state of the art by presenting a new method particularly suitable for identifying, discriminating specifically different groups of microorganisms in a fast, inexpensive and easy to implement.
• the inventors have demonstrated that it is possible to differentiate two groups of microorganisms expressing the same enzymatic activity, by the judicious choice of a combination of substrates specific to the enzymatic activity expressed by the two groups. of microorganisms.
• microorganism covers bacteria, gram positive or gram negative, yeasts and more generally, generally unicellular organisms, invisible to the naked eye, which can be multiplied and manipulated in the laboratory.
• gram-negative bacteria As gram-negative bacteria, mention may be made of the following genera: Pseudomonas, Escherichia, Salmonella, Shigella, Enterobacter, Klebsiella, Serratia, Proteus, Campylobacter, Haemophilus, Morganella, Vibrio, Yersinia, Acinetobacter, Branhamella, Neisseria, Burkholderia , Citrobacter, Hafnia, Edwardsiella, Aeromonas, Moraxella, Pasteurella, Providencia, Actinobacillus, Alcaligenes, Bordetella, Cedecea, Erwinia, Pantoea, Ralstonia, Stenotrophomonas, Xanthomonas and Legionella.
• Gram-positive bacteria mention may be made of the following genera: Enterococcus, Streptococcus, Staphylococcus, Bacillus, Listeria, Clostridium, Gardnerella, Kocuria, Lactococcus, Leuconostoc, Micrococcus, Mycobacteria and Corynebacteria.
• yeasts that may be mentioned include yeasts of the following genera: Candida, Cryptococcus, Rhodotorula, Saccharomyces and Trichosporon.
• reaction medium is meant a medium comprising all the elements necessary for the expression of a metabolism and / or the growth of microorganisms.
• This reaction medium can either serve only as a revelation medium or a culture medium and revelation.
• the culture of the microorganisms is carried out before inoculation and, in the second case, the reaction medium also constitutes the culture medium.
• This medium may contain any other additives such as, for example: peptones, one or more growth factors, carbohydrates, one or more selective agents, buffers, one or more gelling agents, etc.
• This reaction medium may occur in the form of a liquid, ready-to-use gel, that is to say ready for seeding in tube, flask, or on Petri dish.
• enzymatic substrate any substrate that can be hydrolysed by an enzyme into a product for the direct or indirect detection of a microorganism.
• This substrate comprises in particular a first specific part of the enzymatic activity to be revealed and a second part serving as a marker, hereinafter referred to as a marker part.
• This marker part may be chromogenic, fluorogenic, luminescent, etc.
• a chromogenic substrate which is well suited to solid supports (filter, agar, electrophoresis gel), mention may in particular be made of substrates with base of Indoxyl and its derivatives, and substrates based on Hydroxyquinoline or Esculetin and their derivatives, which allow the detection of osidase and esterase activities.
• flavoid derivative is intended to mean, in particular, 3 ', 4'-dihydroxyflavone-4'- ⁇ -D-riboside, 3', 4'-dihydroxyflavone-4'- ⁇ -D-galactoside, 3 ', 4' Dihydroxyflavone-4'- ⁇ -D-glucoside, 3-Hydroxyflavone- ⁇ -D-galactoside, 3-Hydroxyflavone- ⁇ -D-glucoside, 3 ', 4'-Dihydroxyflavone-3', 4'-diacetate.
• Nitrophenol and Nitroaniline substrates and derivatives which can be used to detect osidase and esterase activities in the case of substrates based on nitrophenol, and peptidase activities in the case of substrates based on Nitroaniline. Mention may also be made of coumarin-based substrates and derivatives which also make it possible to detect osidase and esterase activities in the case of substrates based on hydroxycoumarines and in particular 4-methylumbelliferone or cyclohexenesculetin, and peptidase activities in the case of substrates based on aminocoumarines and especially 7-Amino-4-methyl-coumarin.
• Aminophenol substrates and derivatives which make it possible to detect the osidase, esterase and peptidase activities can also be mentioned.
• substrates based on Alizarin and derivatives for detecting osidase and esterase activities There may also be mentioned substrates based on Alizarin and derivatives for detecting osidase and esterase activities.
• Naphtol and Naphtylamine-based substrates and their derivatives, which make it possible to detect the osidase and esterase activities via Naphtol, and the peptidase activities via Naphtylamine, may be mentioned finally.
• Naphtol-based substrate is understood to mean in particular substrates based on ⁇ -Naphthol, ⁇ -Naphtol, 6-Bromo-2-naphthol, Naphtol AS BI, Naphtol AS, p-Naphtolbenzein as defined. in the patent application EP1224196 of the plaintiff.
• This can be substrates of osidase, esterase, phosphatase, sulfatase.
• the osidase substrates are especially substrates of N-acetyl- ⁇ -hexosaminidase, ⁇ -galactosidase, ⁇ -galacotosidase, ⁇ -glucosidase, ⁇ -glucosidase, ⁇ -glucucronidase, ⁇ -cellobiosidase, of ⁇ -mannosidase.
• the enzymatic substrate can also be a natural substrate whose hydrolysis product is detected directly or indirectly.
• a natural substrate mention may in particular be made of Tryptophan for detecting tryptophanase or desaminase activity, a cyclic amino acid (Tryptophan, Phenylalanine, Histidine, Tyrosine) for detecting deaminase activity, Phosphatidyl Inositol for detecting phospholipase activity, etc.
• said same enzymatic activity is chosen from the following enzymatic activities: osidase, esterase, peptidase, and even more preferentially, said same enzymatic activity is chosen from the following enzymatic activities: ⁇ -D-glucosidase, ⁇ -D-galactosidase, alpha -D-glucosidase, alpha-D-galactosidase, alpha-mannosidase, ⁇ -D-glucuronidase, N-acetyl- ⁇ -D-hexosaminidase, ⁇ -D-cellobiosidase, esterase, phosphatase, phospholipase, sulfatase, peptidase.
• the incubation and identification steps are widely known to those skilled in the art.
• the incubation temperature may be 37 ° C.
• it is preferentially aerobic, but it can also be anaerobic, microaerobic or CO 2 .
• the identification can be carried out with the naked eye by visualization of a color change, not not diffusing in the reaction medium, therefore concentrated at the level of the colonies.
• the fluorescence reading devices known to those skilled in the art are used.
• said first group of microorganism is a group of Staphylococcus aureus and said second group is a group of Enterococcus faecalis , said same enzymatic activity is an alpha glucosidase activity, and said first and second substrates are based on Indoxyl.
• the first substrate is 5-Bromo-4-chloro-3-indolyl-N-methyl-alpha-glucoside and the second substrate is 6-chloro-3-indolyl-alpha-glucoside
• said first and second groups of microorganisms are salmonella of different serotypes, said same enzymatic activity is an esterase activity, said first and second substrates are based on Indoxyl.
• said first substrate is 5-Bromo-4-chloro-3-indoxyl-octanoate and said second substrate is 5-Bromo-6-chloro-3-indoxyl-octanoate
• said first group of microorganisms is a group of gram + bacteria and said second group of microorganisms is a group of gram - bacteria; said same enzymatic activity is a beta glucosidase activity; said first substrate is a flavoid derivative and said second substrate is Indoxyl-based.
• said first substrate is 3-Hydroxyflavone-beta-glucoside
• said second substrate is 5-Bromo-4-chloro-N-methyl-3-indolyl-beta-glucoside.
• said first group of microorganisms is a group of gram + bacteria and said second group of microorganisms is a group of gram - bacteria; said same enzymatic activity is beta glucuronidase activity; said first substrate is Naphtol-based and said second substrate is Indoxyl-based.
• said first substrate is p-Naphtholbenzein-beta-glucuronide and said second substrate is 6-chloro-3-indolyl-beta-glucuronide.
• said first group of microorganisms is a group of yeasts and said second group of microorganisms is a group of bacteria; said same enzymatic activity is a hexosaminidase activity; said first substrate is based on Alizarin and said second substrate is based on Indoxyl.
• said first substrate is Alizarin-N-acetyl-beta glucosaminide and said second substrate is 5-Bromo-4-chloro-3-indolyl-N-acetyl-beta-glucosaminide.
• the reaction medium may comprise, in addition, at least one other substrate, preferably several, metabolized by at least one other enzymatic activity, preferably several, the said other enzymatic activity being preferentially selected from ⁇ -D-glucuronidase activity, ⁇ -glucosidase activity, tryptophanase activity, deaminase activity.
• said other substrate is chosen from 6-chloro-3-indolyl-beta-glucuronide, 5-Bromo-4-chloro-3-indolyl-N-methyl- ⁇ -D glucoside, 3 ', 4'-Dihydroxy-4'- ⁇ -D-glucoside, and tryptophan.
• the invention also relates to the use of a reaction medium comprising at least a first enzymatic substrate and at least a second enzymatic substrate, said first and second enzyme substrates being metabolized by the same enzymatic activity, for the identification of a first enzyme substrate. group of microorganisms and a second group of microorganisms, expressing the same enzymatic activity.
• said same enzymatic activity is chosen from the following enzymatic activities: osidase, esterase, peptidase, and even more preferentially, said same enzymatic activity is chosen from the following enzymatic activities: ⁇ -D-glucosidase, ⁇ -D-galactosidase, alpha -D-glucosidase alpha-D-galactosidase, alpha-mannosidase, ⁇ -D-glucuronidase, N-acetyl- ⁇ -D-hexosaminidase, ⁇ -D-cellobiosidase, esterase, phosphatase, phospholipase, sulfatase, peptidase.
• said first group of microorganism is a group of S. aureus and said second group is a group of E. faecalis , said same enzymatic activity is alpha glucosidase activity, and said first and second substrates are based on Indoxyl.
• the first substrate is 5-Bromo-4-chloro-3-indolyl-N-methyl-alpha-glucoside and the second substrate is 6-chloro-3-indolyl-alpha-glucoside.
• said first and second groups of microorganisms are salmonella of different serotypes, said same enzymatic activity is an esterase activity, said first and second substrates are based on Indoxyl.
• said first substrate is 5-Bromo-4-chloro-3-indoxyl-octanoate and said second substrate is 5-Bromo-6-chloro-3-indoxyl-octanoate.
• said first group of microorganisms is a group of gram + bacteria and said second group of microorganisms is a group of gram - bacteria; said same enzymatic activity is a beta glucosidase activity; said first substrate is a flavoid derivative and said second substrate is Indoxyl-based.
• said first substrate is 3-Hydroxyflavone-beta-glucoside
• said second substrate is 5-Bromo-4-chloro-N-methyl-3-indolyl-beta-glucoside.
• said first group of microorganisms is a group of gram + bacteria and said second group of microorganisms is a group of gram - bacteria; said same enzymatic activity is beta glucuronidase activity; said first substrate is Naphtol-based and said second substrate is Indoxyl-based.
• said first substrate is p-Naphtholbenzein-beta-glucuronide and said second substrate is 6-chloro-3-indolyl-beta-glucuronide.
• said first group of microorganisms is a yeast group and said second group of microorganisms is a group of bacteria; said same enzymatic activity is a hexosaminidase activity; said first substrate is based on Alizarin and said second substrate is based on Indoxyl.
• said first substrate is Alizarin-N-acetyl-beta-glucosaminide and said second substrate is 5-Bromo-4-chloro-3-indolyl N-acetyl-beta-glucosaminide.
• the reaction medium may comprise, in addition, at least one other substrate, preferably several, metabolized by at least one other enzymatic activity, preferably several, said other enzymatic activity being preferentially chosen from ⁇ -D-glucuronidase activity, ⁇ -glucosidase activity, tryptophanase activity, deaminase activity.
• said other substrate is chosen from 6-chloro-3-indolyl-beta-glucuronide, 5-bromo-4-chloro-3-indolyl-N-methyl- ⁇ -D-glucoside, 3 ', 4'-Dihydroxy-4'- ⁇ -D-glucoside, and tryptophan.
• the objective is to differentiate at least two groups of microorganisms, for example a first group and a second group, expressing the same enzymatic activity, for example an enzymatic activity of an alpha enzyme.
• each of the two groups of microorganisms is evaluated with respect to different substrates of the alpha enzyme, for example a substrate A, a substrate B, a substrate C.
• Each of the substrates A, B, C is added individually to a reaction medium adapted to the metabolism of said first group and said second group of microorganisms that are to be differentiated.
• a medium A is thus obtained, comprising the substrate A, a medium B comprising the substrate B, a medium C comprising the substrate C ...
• a medium adapted to yeast metabolism may be Sabouraud medium possibly partially or totally lacking glucose.
• a medium adapted to the metabolism of the bacteria may be in particular a Trypcase Soy medium or a Columbia medium.
• a medium adapted to the metabolism of urinary microorganisms can be in particular a CPS ID 3 medium, free of its enzymatic substrates.
• reaction media may be liquid media or gelled media.
• Each medium is aliquoted.
• One or more strains of each of the groups of microorganisms to be differentiated is seeded on an aliquot of each medium. These cultures are then incubated under the appropriate conditions.
• each of the substrates is evaluated, possibly after different incubation times, so as to determine if there is between at least two substrates of the same enzymatic activity, a hydrolysis differential linked to the group of microorganisms. .
• an analogous reaction medium supplemented with enzymatic substrates having this hydrolysis differential is produced.
• he is aliquoted.
• One or more strains of each of the groups of microorganisms to be differentiated is seeded on an aliquot of this medium.
• These cultures are then incubated under the appropriate conditions and then examined, possibly after different incubation times, to evaluate whether the enzymatic expression differential makes it possible to differentiate the groups of microorganisms. studied.
• the differentiation of the groups depends not only on the hydrolysis differential between the substrates, but also on the difference between the signals produced by the hydrolysis of each of the substrates and any interactions, especially at the level of the enzyme substrates and / or signals produced.
• the example previously developed can be implemented in a similar manner to discriminate not two groups of microorganisms, but 3, 4 or more groups of microorganisms.
• Example 2 Use of two substrates of alpha-glucosidase based on Indoxyl 5-Bromo-4-chloro-3-indolyl-N-methyl-alpha-glucoside (XN-Me- ⁇ -GLU) in combination with the 6 -Chloro-3-indolyl-alpha-glucoside (Rose- ⁇ -GLU) to discriminate the species Staphylococcus aureus and Enterococcus faecalis
• the media thus formed were distributed in Petri dishes.
• this medium is particularly suitable for the detection of gram-positive cocci resistant to glycopeptides, especially those having a so-called acquired resistance, such as Vancomycin - resistant Staphylococcus aureus (VRSA) or Enterococcus faecalis or Vancomycin- resistant E. faecium (VRE).
• VRSA Vancomycin - resistant Staphylococcus aureus
• Enterococcus faecalis Vancomycin- resistant E. faecium
• Example 3 Use of two Escherase substrates based on Indoxyl 5-Bromo-4-chloro-3-indoxyl-octanoate (X-C8) in combination with 5-Bromo-6-chloro-3-indoxyl octanoate (Magenta-C8) to discriminate salmonella strains of different serotypes
• the media thus formed were distributed in Petri dishes.
• EXAMPLE 4 Use of a Beta-Glucosidase 3-Hydroxyflavone-Beta-Glucoside (HF- ⁇ -GLU) or 3 ', 4'-Di-Hydroxyflavone-Beta-Glucoside Substrate (DHF- ⁇ -GLU) with Indoxyl-based substrate 5-Bromo-4-chloro-N-methyl-3-indolyl-beta-glucoside (XN-Me- ⁇ -GLU) to discriminate gram + bacteria and gram-positive bacteria with beta-glucosidase activity
• the media thus formed were distributed in Petri dishes. Strains of Enterobacter cloacae, Klebsiella pneumoniae, Serratia marcescens, Enterococcus faecium , E. faecalis , Enterococcus gallinarum , S. aureus , E. coli , were seeded, at the calibrated level of 10 ⁇ l, on each medium, starting from Calibrated suspensions of 0.5 McFarland. All cultures were incubated 24 h at 37 ° C.
• Example 5 Use of a substrate of p-Naphtholbenzein-beta-glucuronide (pNB- ⁇ -GUR) with a substrate based on indoxyl 6-Chloro-3-indolyl-beta-glucuronide (Rose- ⁇ -GUR) to discriminate gram + and gram-positive bacteria with beta-glucuronidase activity
• pNB- ⁇ -GUR p-Naphtholbenzein-beta-glucuronide
• Rose- ⁇ -GUR indoxyl 6-Chloro-3-indolyl-beta-glucuronide
• the media thus formed were distributed in Petri dishes.
• EXAMPLE 6 Use of an Alizarin-Based Substrate, Alizarin-beta-N-acetylglucosaminide (Aliz- ⁇ -NAG) with a 5-Bromo-4-chloro-3-indolyl Indoxyl Based Substrate -beta-N-acetylglucosaminide (X- ⁇ -NAG) for discriminating bacteria and yeasts having hexosaminidase activity
• the media thus formed were distributed in Petri dishes. Different strains of microorganisms were seeded, at the calibrated level of 10 .mu.l, on each medium, from calibrated 0.5 McFarland suspensions. All cultures were incubated 24 h at 37 ° C.
• Table 10 show that the positive hexosaminidase bacteria preferentially hydrolyze the Alizarin-based substrate while the positive hexosaminidase yeasts preferentially hydrolyze the Indoxyl-based substrates. As a result, it is possible to separate yeast bacteria having the same enzymatic activity, by using two substrates for this same activity, the affinity of the enzyme of each group is different.

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